Endogenous regulator of RNA silencing in plants

ABSTRACT

Compositions and methods are described which use RAV proteins and genes that encode such proteins to regulate RNA silencing in plant cells. Novel ntRAV proteins and genes are also described.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant No.5R01GM061014-04 awarded by the U.S., National Institutes of Health ofthe Department of Health and Human Services. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to methods of modulating gene expressionin plants and to novel proteins and nucleotide sequences, and inparticular, to methods of using such novel proteins and nucleotides forregulating the effects of post transcriptional gene silencing in plants.

(2) Description of the Related Art

With the advent of recombinant DNA technology in the 1970s, the geneticmanipulation of plants entered a new age. Genes and traits previouslyunavailable through traditional breeding became available through DNArecombination, and with greater specificity than ever before.Commercially important genes from sexually incompatible plants, animals,bacteria or insects can now be successfully introduced into plants.Products of modern plant genetic engineering are already on the market,and examples include such transgenic plants as slow-softening tomatoes,cotton and corn plants resistant to herbicides and insects, and soybeanswith altered fatty acid profiles. With many more products in thepipeline, the genetic engineering of plants is expected to have aprofound impact on the future of agriculture.

Modern plant genetic engineering can involve the transfer of a desiredgene into the plant genome and regeneration of a whole plant from thetransformed tissue. Unfortunately, many of these efforts have been metwith mixed results with regards to predictable and sustainable levels ofgene expression.

Many factors affect gene expression in plants. One mechanism, RNAsilencing, is an important regulatory mechanism that has only recentlybeen intensively studied. RNA silencing is characterized by reducedlevels of the specific mRNA encoded by the silenced gene. Individualcases of silencing include those in which mRNA level is regulatedtranscriptionally and those in which it is regulatedpost-transcriptionally.

RNA silencing in transgenic plants was first discovered after genetictransformation of plant tissues with multiple copies of an exogenoustransgene that shared some homology with an endogenous gene. Researchersnoticed that over time, high levels of exogenous gene expression wouldunexplainably falter and drop to low levels.

Recently, it has been reported that RNA silencing can be induced byplant virus infections in the absence of any known homology between theviral genome and an endogenous gene. It has been proposed that genesilencing may have evolved as a defense mechanism against viralinvasion.

All genomes, including those of plants, animals, and fungi have specificdefense mechanisms against infection by foreign bioactive agents. Thus,it has been proposed that RNA silencing is an ancestral mechanism thatplant cells use as a defense against invading nucleotide molecules in asequence specific manner. Therefore, one emerging view is that RNAsilencing is part of a sophisticated network of interconnected pathwaysfor cellular defenses (e.g., against viruses and transposons), RNAsurveillance, and development, and that it could become a powerful toolto manipulate gene expression.

When viruses or transgenes are introduced into plants, they oftentrigger a particular host response that is generally referred to aspost-transcriptional gene silencing (PTGS) or cosuppression. Themechanism of PTGS is believed to be similar to that of RNA interference(RNAi) in animals. The process appears to be initiated bydouble-stranded RNA (dsRNA) molecules, which may be generated byreplicative intermediates of viral RNAs or by aberrant transgene-codedRNAs (these RNA molecules may become double-stranded when copied and/oramplified by an RNA-dependent RNA polymerase). The dsRNAs are digestedinto 21-23 nucleotide (nt) small interfering RNAs (siRNA). Evidenceindicates that siRNAs are produced when the enzyme Dicer, a member ofthe RNase III family of dsRNA-specific ribonucleases, digests dsRNA inan ATP-dependent, processive manner. Successive cleavage events degradethe RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3′overhangs. The conversion of dsRNA into siRNAs requires additionalprotein co-factors that may recruit the dsRNA to Dicer or stabilize thesiRNA.

These siRNAs direct the degradation of target mRNAs complementary to thesiRNA sequence by subsequently binding to a nuclease complex to formwhat is known as the RNA-induced silencing complex (RISC). Thereafter,the RISC complex binds and destroys the complementary target mRNAs,which in turn, leads to the silenced target RNA phenotype.

As mentioned previously, in both plants and animals, RNA silencing hasevolved, at least in part, as an antiviral defense pathway. In response,many viruses have developed genes that encode suppressors of silencing.For example, it has been reported that certain plant viruses encodeproteins that can suppress RNA silencing. One suppressor protein inparticular, helper component-proteinase (HC-Pro), is produced by anumber of plant potyviruses. HC-Pro acts to suppress transgene-inducedsilencing by interfering with the accumulation of the 21-23 nucleotidesiRNAs. It has been found that infection of transgenic plants by virusesexpressing HC-Pro can cause wholesale suppression of RNA silencing,resulting in the stabilization of target gene expression. For severalreasons, however, it is not desirable to use the viral HC-Pro protein asa vector to suppress silencing. Furthermore, in cases where suppressionof silencing is undesirable, actual resistance to HC-Pro suppression isrequired.

These recent studies on RNA silencing have furthered the understandingof the regulatory mechanisms underlying gene expression. However, Inorder to better utilize transgenic plants, the mechanisms behindtransgene expression and endogenous gene suppression need to becontrollable. The ability to quickly and easily create knock outphenotypes using protein components of the RNA silencing pathway wouldbe desirable in terms of plant biotechnology.

From the foregoing, it can be seen that a need exists for improvedmethods of modulating gene expression in plant cells and plants, and inparticular, for methods of regulating—by either reducing orenhancing—the expression of a certain target genes in plants. Suchmethods are needed to control gene suppression and to obtain acceptableexpression levels of genes of interest.

SUMMARY

Briefly therefore, the present invention is directed to a novel methodof regulating gene silencing in a plant cell, the method comprisingmodulating in the plant cell the amount of and/or the activity of a RAVprotein.

The present invention is also directed to a novel method of regulatingpost transcriptional gene silencing in a plant, the method comprising:controlling in the plant the expression of a gene that encodes a RAVprotein; or controlling in the plant the amount of and/or the activityof a RAV protein.

The present invention is also directed to a novel isolated nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, or asequence that is substantially identical thereto.

The present invention is also directed to a novel chimeric genecomprising the nucleotide sequence of SEQ ID NO:1, or a sequence that issubstantially identical thereto, operably linked to suitable regulatorysequences.

The present invention is also directed to a novel recombinant vector fortransformation of plant cells, the vector comprising a polynucleotidehaving the sequence of SEQ ID NO:1, or a sequence that is substantiallyidentical thereto.

The present invention is also directed to a novel plant cell comprisingan introduced nucleotide sequence according to SEQ ID NO:1, or asequence that is substantially identical thereto.

The present invention is also directed to a novel plant comprising anintroduced nucleotide sequence according to SEQ ID NO:1, or a sequencethat is substantially identical thereto.

The present invention is also directed to a novel plant seed comprisingan introduced nucleotide sequence according to SEQ ID NO:1, or asequence that is substantially identical thereto.

The present invention is also directed to a novel isolated proteincomprising the amino acid sequence of SEQ ID NO:2, or a sequence that issubstantially identical thereto.

The present invention is also directed to a novel antibody that bindswith a protein comprising the amino acid sequence of SEQ ID NO:2, or asequence that is substantially identical thereto, or binds with apolynucleotide comprising the nucleic acid sequence of SEQ ID NO:1, or asequence that is substantially identical thereto.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of novel proteins andpolynucleotides and to methods which use the novel proteins andpolynucleotides to control RNA silencing of a target gene in a host celland thereby to effectively modulate expression of the target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs of Northern Blot panels that indicate thelevels of ntRAV in samples taken from leaves of wild type tobacco plants(WT), from tobacco plants that overexpressed ntRAV with a 35S promoter(ntRAV), and from the knockdown line of tobacco plants having reducedlevels of ntRAV expression (dsRAV), at 24, 30 and 37 days postgermination (P.G.);

FIG. 2 illustrates the developmental silencing of a sense transgeneencoding the reporter enzyme GUS in the tobacco transgenic line 6b5,wherein silencing is seen to occur developmentally through days 26, 28and 32 P.G. and is seen to begin in the vascular system and spreadsthroughout the leaf; and also shown on the right is the progression ofsilencing in an adult plant from the bottom of the plant towards thetop;

FIG. 3 shows the effect of reducing ntRAV expression on sense transgenesilencing of the reporter gene GUS in tobacco transgenic line 6b5.Histochemical staining to localize GUS expression in plants at 24 daysafter germination is shown in the upper part of the figure. Shown at thefar left is a leaf from the 6b5 transgenic plant that also expressesHC-Pro, a viral suppressor of RNA silencing (HC-Pro X 6b5). This leaf isdark colored throughout because silencing of the GUS gene is blocked.The second leaf from the left shows a leaf from a 6b5 transgenic plantthat also over-expresses the ntRAV gene and is also suppressed for RNAsilencing (ntRAV X 6b5). The third leaf from the left is from the 6b5line in an otherwise wildtype background (WT X 6b5). This leaf shows theinitiation of GUS silencing in the veins of the leaf: thus, the veins ofthe plants are not dark colored in some places due to lack of GUSactivity. The last leaf on the right is from the 6b5 line that alsocarries a transgene that expresses double-stranded (ds) RNA for theendogenous ntRAV gene (dsRAV X 6b5). This leaf shows more extensivesilencing of the GUS transgene as indicated by more extensive loss ofcolor in the veins. Molecular data from the same plants is shown in thelower part of the figure. The level of GUS mRNA and GUS siRNAs is shownin the sense-transgene silenced 6b5 transgenic line at 7, 14 or 24 dayspost-germination in an otherwise wildtype background (wt) or in thepresence of the HC-Pro transgene (HC-Pro) or a transgene that producesdouble-stranded ntRAV RNA (dsRAV) and thereby induces silencing of thentRAV gene. In an otherwise WT background, GUS silencing in line 6b5begins between 14 and 24 days after germination as indicated by thereduced level of GUS mRNA and the simultaneous appearance of the GUSsiRNAs, which are diagnostic of RNA silencing. HC-Pro blocks the onsetof silencing and prevents siRNA accumulation. In contrast, the presenceof the dsRAV transgene accelerates GUS silencing as indicated by thereduced levels of GUS mRNA even at 7 days after germination and theappearance of GUS siRNAs by 14 days after germination;

FIG. 4 illustrates the effectiveness of RNA silencing in tobacco plants,and contrasts silencing in a wild type 6b5 cross (wt x 6b5) withsilencing in a knockdown ntRAV plant (dsRAV x 6b5), showing thatsilencing proceeds from the bottom to the top of the plant, and isvisually more advanced in the knockdown ntRAV plant than in the wildtype plant;

FIG. 5 shows a comparison of the percent amino acid similarity betweenntRAV and several RAV proteins from Arabidopsis thaliana, andRAV-related proteins from pepper and rice;

FIG. 6 shows a comparison of the amino acid sequences of ntRAV versussix RAV proteins from Arabidopsis thaliana;

FIG. 7 shows a comparison of the amino acid sequences of ntRAV versusfive RAV-related proteins from rice;

FIG. 8 shows a comparison of the amino acid sequences of ntRAV versus aRAV-related protein from pepper;

FIG. 9 shows a GUS-silenced L-1 line of Arabidopsis sp. plant havingwild-type RAV-2 gene on the left and on the right, a GUS-silenced L-1line of Arabidopsis sp. plant having a knockout of its RAV-2 gene by aT-DNA insertion, and illustrating the enhancement of sense transgenesilencing in the knockout plant by more extensive reduction in theexpression of GUS as indicated by its lighter colored leaves; and

FIG. 10 shows a comparison of the amino acid sequences of the AP2 and B3DNA binding sites of ntRAV with the amino acid sequences of comparablesites of other RAV proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has been discovered thatRNA post transcriptional gene silencing in plants can be regulated bymodulating in the plant cell the amount of and/or the activity of amember of the RAV family of proteins, which is described below indetail. One such RAV protein includes an amino acid sequence accordingto SEQ ID NO: 2, and has been shown to act an endogenous suppressor ofpost transcriptional gene silencing. The RAV protein having SEQ ID NO:2is encoded for by a nucleotide sequence of SEQ ID NO:1. Because thisprotein was originally isolated from tobacco (Nicotiniana tabacum), anddue to its similarity with proteins of the RAV family of Arabidopsisthaliana, the protein that includes the amino acid sequence of SEQ IDNO:2 is referred to herein as “ntRAV”.

The term “modulating”, as used herein in relation to affecting theamount of a protein in a plant cell, means taking action that increasesthe amount of the protein in the cell or taking action that decreasesthe amount of the protein in the cell. As used herein in relation toaffecting the activity of a protein in a plant cell, the term“modulating” means taking action that increases the biological activityof the protein in the plant cell or taking action that decreases thebiological activity of the protein in the cell. It is preferred that anyaction that is taken, within the meaning of the present invention, isone that permits the plant cell to continue to live and function.

In particular, it has been discovered that when a RAV protein isoverexpressed in plants, it delays the onset of RNA silencing, thusallowing longer expression of a normally silenced target gene.Conversely, when the amount of or the expression of this same protein isdecreased by various means, the target genes are silenced more rapidlyand more effectively. Moreover, when the amount of or the expression ofthis protein is decreased, the plant can be made to be less susceptibleto the RNA silencing-suppressing effects of plant viruses expressingproteins such as HC-Pro.

It has been shown that by modulating the amount of and/or the activityof ntRAV protein in a plant cell, one can regulate the RNA silencingmechanism of the cell. For example, by reducing the amount of ntRAVprotein in the cell, the expression of deleterious genes can be turnedoff. Historically, such silencing has often been incomplete. Thus,knocking out the expression of ntRAV protein is a useful way to increasethe effectiveness of RNA silencing in cases where such upregulation isdesirable. Conversely, in some instances, RNA silencing limitsbiotechnologies aimed at high level gene expression in plants. Thus,overexpression of ntRAV protein is useful in delaying/suppressingsilencing in situations where that is the desired outcome. In the lattercase, the ntRAV protein might be useful in conjunction with other viraland endogenous suppressors of silencing, such as HC-Pro, or rgs-CaM, forexample, to enhance or reduce their effectiveness. For additionalinformation about the activity of rgs-CaM, see Anandalakshmi, R. et al.,Science, 290:142-144 (2000).

When ntRAV is over-expressed in tobacco, the plants display a delay inthe onset of RNA silencing induced by a sense transgene. When expressionof the ntRAV protein is knocked down using an RNAi construct, the onsetof sense transgene silencing is accelerated. Furthermore, more effectivesilencing occurs in adult plants when the expression of this protein isreduced. In addition, knockout of a related gene in Arabidopsis thalianausing a T-DNA insertion into the gene also enhances sense transgenesilencing. These results suggest that the ntRAV protein that isdescribed herein, and substantially identical proteins, are strongendogenous negative regulators (suppressors) of RNA silencing. Thus,genetic manipulation of the expression of the gene encoding thisprotein, and/or modulation of the level of the ntRAV protein itself,provides a mechanism to alter RNA silencing—either to suppress it orenhance it.

RNA silencing is frequently used to reduce the expression of deleteriousgenes in plants. However, many plant viruses encode proteins thatsuppress RNA silencing. Thus, infection by certain plant virusespotentially endangers the RNA silencing in engineered plants. Inparticular, the potyviruses encode a potent suppressor of RNA silencingthat is capable of reversing silencing even when it has already beenestablished in the plant. The RAV-related proteins that are the subjectof this patent are, in part, defined by their interaction with HC-Proand HC-Pro may well suppress RNA silencing via alterations in RAVactivity. When the expression of RAV-related proteins is reduced orknocked out in either tobacco or Arabidopsis, RNA silencing is enhanced(i.e. it occurs faster and to a greater extent than in wildtype plants).The enhanced silencing is beneficial in cases where silencing isdesired. In addition, it is possible that plants in which expression oractivity of RAV-related proteins has been altered may be resistant toHC-Pro suppression of RNA silencing. Thus, if HC-Pro suppressessilencing via alterations in RAV-related activities, then it would notbe able to suppress silencing in RAV knock-out plants. Because thepotyviruses that encode HC-Pro are common, HC-Pro resistant RNAsilencing would be very beneficial in cases where RNA silencing isdesired.

In summary, it has been found that overexpression of ntRAV delays theonset of silencing in transgenic plants (e.g., tobacco). Knockdown ofexpression of the protein in tobacco enhances silencing, allowingsilencing to occur more rapidly and more effectively. Knockout of arelated transcription factor in Arabidopsis also enhances RNA silencing.There is no developmental phenotype associated with altered expressionof the ntRAV protein. It is also believed that knockout of the geneencoding this protein may eliminate the ability of the viral suppressorof silencing termed HC-Pro to block RNA silencing.

The present invention includes, in one embodiment, a novel protein(i.e., ntRAV), which interacts with a plant viral suppressor ofsilencing called HC-Pro in a yeast two-hybrid system. As mentionedabove, ntRAV comprises the amino acid sequence of SEQ ID NO.2, which iscoded for by the polynucleotide sequence of SEQ ID NO.1. Relatedproteins, several of which are described below, have been identifiedthat share the characteristic of utility for the regulation of genesilencing in plants, and they are included in the scope of an embodimentof the present invention.

ntRAV is a member of a group of related proteins. These proteins aredistinguished by the presence of two distinct DNA binding motifs calledAP2 and B3. This is a unique feature of the RAV and RAV-like proteins,See Kagaya, Y. et al., Nucleic Acids Res., 27:470-478 (1999). Other DNAbinding proteins commonly have one DNA binding motif or one or morecopies of the same DNA binding motif. Only the RAVs have two distincttypes of DNA binding motifs and this feature distinguishes them from allother proteins. Further description of the AP2 binding domain can befound in Jofuku, K. D. et al., Plant Cell, 6:1211-1225 (1994), andOhme-Takagi, M. et al., Plant Cell, 7:173-182 (1995). Furtherinformation about the B3 binding domain can be found in Giraudat, J. etal., Plant Cell, 4:1251-1261 (1992), and McCarty, D. R. et al., Cell,66:895-905 (1991). FIG. 18 shows a comparison of the AP2 and B3 DNAbinding motif sequences of ntRAV with those from related proteins inpepper (csRAV), Arabidopsis (six proteins with At numbers) and rice(five proteins starting with “gi”). The percent similarity of eachprotein sequence with ntRAV is indicated after the name of the protein.The similarity data for the two DNA binding motifs along with theoverall similarity of the whole protein to ntRAV is presented in thetable shown in FIG. 5.

As used herein, ntRAV and all RAV and RAV-like proteins will be includedin the terms “RAV protein”. Likewise, genes that encode ntRAV and anyRAV and RAV-like proteins will be included in the terms “RAV gene”, or“gene encoding a RAV protein”, or the like. A RAV protein, as definedherein, is a protein having both AP2 and B3 DNA binding domains. When itis said that a RAV protein has an AP2 DNA binding domain, it is meantthat the protein contains an amino acid sequence that has at least a 50%similarity to the amino acid sequence between amino acids 63 and 119 ofntRAV. It is preferred that the RAV protein has an amino acid sequencethat has at least a 60% similarity to the amino acid sequence betweenamino acids 63 and 119 of ntRAV, more preferred is a similarity of atleast 70%, and yet more preferred is a similarity of at least 80%. Themeaning of similarity between respective amino acid sequences andnucleic acid sequences is explained below.

The amino acid sequence of the AP2 binding domain of ntRAV is shown inSEQ ID NO:27, and includes amino acids 63-119. Comparable AP2 bindingdomains on other RAV proteins are shown in FIG. 10, and include AP2binding domains for pepper (csRAV, SEQ ID NO:28), six RAV proteins fromArabidopsis thaliana (At proteins, SEQ ID NOS:29-34), and five rice RAVproteins (gi proteins, SEQ ID NOS:35-39). Percent similarity is shownfor each sequence as compared to the AP2 binding site of ntRAV.

When it is said that a RAV protein has a B3 DNA binding domain, it ismeant that the protein contains an amino acid sequence that has at leasta 40% similarity to the amino acid sequence between amino acids 197 and256 of ntRAV (with numbering starting from the amino end). It ispreferred that the RAV protein has an amino acid sequence that has atleast a 60% similarity to the amino acid sequence between amino acids197 and 256 of ntRAV, more preferred is a similarity of at least 70%,and yet more preferred is a similarity of at least 80%.

The amino acid sequence of the B3 binding domain of ntRAV is shown inSEQ ID NO:40, and includes amino acids 197-256. Comparable B3 bindingdomains on other RAV proteins are shown in FIG. 10, and include B3binding domains for pepper (csRAV, SEQ ID NO:41), six RAV proteins fromArabidopsis thaliana (At proteins, SEQ ID NOS:42-47), and five rice RAVproteins (gi proteins, SEQ ID NOS:48-52). Percent similarity is shownfor each sequence as compared to the B3 binding site of ntRAV.

In some embodiments of the present invention, polynucleotides thatcomprise nucleotide sequences which encode for the respective AP2 and B3DNA binding domains of the RAV proteins are to be included in the scopeof the invention. Because the nucleotide sequences of the genes encodingntRAV and RAV proteins are known, identification of a RAV protein havingan amino acid sequence that is within a defined range of similarity toan ntRAV AP2 or B3 binding site would also be considered to identify thecorresponding nucleic acid that encodes for that protein. Thus, thepolynucleotide encoding the RAV protein would be within the same rangeof similarity as expressed for the protein when compared with thenucleotide sequence that encodes the reference ntRAV protein AP2 or B3binding site. For example, identification of a RAV protein having anamino acid sequence with 80% similarity to the AP2 site of ntRAV and 70%similarity to the B3 site of ntRAV, would also identify thepolynucleotide that encoded the RAV protein, which would have 80%nucleic acid similarity to the nucleotide sequence encoding for the AP2site of ntRAV and 70% similarity to the nucleotide sequence encoding forthe B3 site of ntRAV.

The amino acid (polypeptide) sequence of Tobacco (Nicotiana tabacum)ntRAV is shown in SEQ ID NO:2. The amino acid sequences of six RAVtranscription factors of Arabidopsis thaliana are shown as SEQ IDNOS:3-8. The amino acid sequences of a pepper RAV transcription factoris shown in SEQ ID NO:9, and the amino acid sequences of five RAVproteins from rice are shown as SEQ ID NOS:10-14. It is believed thatall of the proteins that include the amino acid sequences shown in SEQID NOS:2-14 are members of the RAV family of proteins.

A comparison of the amino acid sequence similarity of ntRAV with severalRAV transcription factors of Arabidopsis, and with RAV-related proteinsof pepper and rice is shown in FIG. 5. It can be seen that amino acidpercent sequence identity ranges from 48.6% to 86.8%.

FIG. 6 shows a comparison of the amino acid sequence of ntRAV withseveral Arabidopsis RAV transcription factors. The same type ofcomparison is shown in FIG. 7 for rice RAV-related proteins, and in FIG.8 for pepper RAV-related proteins.

The polynucleotide sequence that encodes ntRAV of tobacco is shown asSEQ ID NO:1. In addition, SEQ ID NOS: 15-26 show polynucleotidesequences that encode RAV proteins of Arabidopsis, pepper, and rice,respectively.

While not wishing to be bound by this or any other particular theory, itis believed that the ntRAV/HC-Pro interaction is indicative of ntRAVhaving a direct role in the HC-Pro suppression of silencing. Forexample, to determine if ntRAV plays a role in RNA silencing, wild typeplants and plants overexpressing ntRAV were crossed to a tobaccotransgenic line containing a silenced GUS sense-transgene, and theprogeny were analyzed for the expression of the ntRAV and GUS genes. Theendogenous ntRAV gene is expressed at high levels in seedlings and thendecreases precipitously at about 3 weeks, the same time that silencingof the GUS sense-transgene initiates. The expression of high levels ofntRAV is extended by about 2 weeks in the ntRAV overexpressing line, andthe onset of RNA silencing is delayed correspondingly. A high level ofntRAV is therefore correlated with inability of plants to initiatesilencing of GUS sense-transgenes. This result raises the possibilitythat the RAV proteins directly or indirectly control expression oractivity of components of the silencing mechanism.

The invention overcomes the limitations of the prior art by providingmethods and compositions for modulating silencing of gene expression inplants, and thereby modulating gene expression itself. In particular,the present invention encompasses a novel protein (i.e., ntRAV, andproteins that are substantially identical) and a novel method of using afamily of proteins (i.e., RAV proteins) that are related to theArabidopsis thaliana RAV family, and which are capable of modulating RNAsilencing of particular coding sequences, even when provided in theabsence of viral factors.

The present invention is useful for modulating the expression of one ormore target genes by modulating RNA silencing. The present invention canaccomplish such modulation by overexpressing or knockout of one or moreof the endogenous RAV sequences described herein. For example,overexpression of one or more of the RAV sequences leads to suppressedRNA silencing and therefore, enhanced target gene expression.Conversely, reduced expression of one or more of the endogenous RAVsequences leads to enhanced RNA silencing and therefore, suppressedtarget gene expression.

Therefore, in one embodiment, the present invention may find particularuse for enhancing expression of one or more transgenes by suppressingRNA silencing through overexpression of one or more of the RAV proteinsdescribed herein, as silencing of transgenes can frequently occur,especially when transgenes are present in more than one copy in agenome. This affect may be achieved without the need for fusions betweena transgene coding sequence and one of the RAV sequences describedherein, or alternatively, using such a fusion.

In another embodiment, the present invention may find particular use forsuppressing the expression of one or more transgenes by enhancing RNAsilencing through the reduction of expression of one or more of theendogenous RAV proteins described herein. Likewise, this affect may beachieved without the need for fusions between a transgene codingsequence and one of the RAV proteins described herein, or alternatively,using such a fusion.

Compositions and methods for modulating the expression of a targetsequence in a plant are provided. That is, the expression of the targetsequence may be enhanced or decreased. In preferred embodiments, areduced expression of the target sequence is effected.

The target sequence comprises any sequence of interest, including genes,regulatory sequences, and the like. Genes of interest include thoseencoding agronomic traits, insect resistance, disease resistance,herbicide resistance, sterility, grain characteristics, and the like.The genes may be involved in metabolism of oil, starch, carbohydrates,nutrients, and the like. Genes or traits of interest include, but arenot limited to, environmental- or stress-related traits, disease-relatedtraits, and traits affecting agronomic performance. Target sequencesalso include genes responsible for the synthesis of proteins, peptides,fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates,flavors, odors, toxins, carotenoids, hormones, polymers, flavonoids,storage proteins, phenolic acids, alkaloids, lignins, tannins,celluloses, glycoproteins, glycolipids, and the like.

The invention relates to the modulation of RNA silencing of a targetgene, and in preferred embodiments, the enhancement of RNA silencing ofa target gene in plants. The terms “RNA silencing of a target gene” aregenerally used to refer to suppression of expression of a gene. Thedegree of reduction may be partial or total reduction in production ofthe encoded gene product. Therefore, these terms should not be taken torequire complete “silencing” of expression of a gene.

The methods and compositions of the invention are useful in anysituation where modulation of the expression of a nucleotide sequence ina plant cell is desired. Thus, the methods are useful for modulating theexpression of endogenous as well as exogenous sequences. For example,for exogenous sequences, the endogenous RAV sequences described hereincan be overproduced or RAV can be added in order to enhance expressionof genes normally silenced by transgene-induced gene silencing. In otherexamples, expression of one or more of the RAV sequences may be blockedor reduced, or the activity or amount of the RAV protein can byinhibited or reduced in order to more effectively suppress expression ofa target gene that is normally silenced. Therefore, the target sequencemay be any nucleotide sequence of interest.

Overexpression of RAV in a plant cell can be accomplished by operablylinking one or more of the RAV sequences described herein to anyexogenous promoter that would allow for stronger expression that the RAVendogenous promoter. For example, operably linking a RAV sequence to acauliflower mosaic virus 35S promoter in a suitable expression cassetteand then inserting such an expression cassette into a plant cell couldgive overexpression of ntRAV transcripts and/or RAV polypeptides.

Suppression of endogenous RAV expression and commensurate reduction of atarget gene's expression can be accomplished by creating a “knock-out”or “disruption” of one or more of the endogenous RAV sequencesidentified herein and then allowing homologous recombination between theendogenous RAV gene and the disrupted knock-out nucleic acid sequence. Aknock-out or disrupted RAV sequence can be prepared by inserting a“non-RAV” sequence within the endogenous RAV sequence or by rearrangingor deleting parts of the RAV sequence and then inserting the disruptedRAV sequence into a host cell. Suitable non-RAV sequences that couldknock-out the endogenous RAV expression could be selectable markers orany other such markers.

As used herein, the term “DNA” means deoxyribonucleic acid. As usedherein, the term “cDNA” means complementary deoxyribonucleic acid.

As used herein, the terms “coding sequence” mean a polynucleotidesequence that is translated in an organism to produce a protein. Theterm “coding DNA sequence” is a DNA sequence from which the informationfor making a peptide molecule, mRNA or tRNA are transcribed. A DNAsequence may be a gene, combination of genes, or a gene fragment.

As used herein, “complementary” polynucleotides are polynucleotides thatare capable of base-pairing according to the standard Watson-Crickcomplementarity rules. Specifically, purines will base pair withpyrimidines to form combinations of guanine paired with cytosine (G:C)and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. Two polynucleotidesmay hybridize to each other if they are complementary to each other, orif each has at least one region that is substantially complementary tothe other.

As used herein, the term “gene” should be understood to refer to a unitof heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain itself, which has that sequence of nucleotides. The term“sequence” is used in the similar way in referring to RNA chains, linearchains made of ribonucleotides. The gene may include regulatory andcontrol sequences, sequences which can be transcribed into an RNAmolecule, and may contain sequences with unknown function. The majorityof the RNA transcription products are messenger RNAs (mRNAs), whichinclude sequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent strains of the same type of organism, or even within aparticular strain of the organism, without altering the identity of thegene.

As used herein, the term “heterologous” is used to indicate arecombinant DNA sequence in which the DNA sequence and the associatedDNA sequence are isolated from organisms of different species or genera.

A polynucleotide may be “introduced” or “transformed” into any cell byany means known to those of skill in the art, including transfection,transformation or transduction, transposable elements, electroporation,Agrobacterium infection, polyethylene glycol-mediated uptake, particlebombardment, and the like. The introduced polynucleotide may bemaintained in the cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the genome of thehost cell. Alternatively, the introduced polynucleotide may be presenton an extra-chromosomal non-replicating vector and be transientlyexpressed or transiently active. As used herein, the term “transform” or“introduce” refers to the introduction of a polynucleotide (single ordouble stranded DNA, RNA, or a combination thereof) into a living cellby any means. Transformed cells, tissues and plants encompass not onlythe end product of a transformation process, but also the progenythereof, which retain the polynucleotide of interest.

As used herein, the term “isolated polynucleotide” is a polynucleotidethat is substantially free of the nucleic acid sequences that normallyflank the polynucleotide in its naturally occurring replicon. Forexample, a cloned polynucleotide is considered isolated. Alternatively,a polynucleotide is considered isolated if it has been altered by humanintervention, or placed in a locus or location that is not its naturalsite, or if it is introduced into a cell by agroinfection.

As used herein, a “normal” or “endogenous” form of a gene (wild type) isa form commonly found in natural populations of an organism. Commonly asingle form of a gene will predominate in natural populations. Ingeneral, such a gene is suitable as a normal form of a gene; however,other forms which provide similar functional characteristics may also beused as a normal gene. In particular, a normal form of a gene does notconfer a growth conditional phenotype on the organism having that gene,while a mutant form of a gene suitable for use in these methods canprovide such a growth conditional phenotype.

As used herein, “nucleic acid” and “polynucleotide” refer to RNA or DNAthat is linear or branched, single or double stranded, or a hybridthereof. The polynucleotides of the invention may be produced by anymeans, including genomic preparations, cDNA preparations, in vitrosynthesis, RT-PCR and in vitro or in vivo transcription. The term“polynucleotide” means a chain of at least two nucleotides joinedtogether. The chain may be linear, branched, circular or combinationsthereof. Nucleotides are generally those molecules selected forbase-pairing and include such molecules as guanine, cytosine, adenine,uracil, and thymine.

As used herein, the term “operatively linked to” or “associated with”means two DNA sequences which are related physically or functionally.For example, a promoter or regulatory DNA sequence is said to be“associated with” a DNA sequence that codes for an RNA or a protein ifthe two sequences are operatively linked, or situated such that theregulator DNA sequence will affect the expression level of the coding orstructural DNA sequence.

As used herein, the term “plant” refers to whole plants, plant organsand tissues (e.g., stems, roots, ovules, fruit, stamens, leaves,embryos, meristematic regions, callus tissue, gametophytes, sporophytes,pollen, microspores and the like), seeds, plant cells and the progenythereof.

As used herein, the term “plant tissue” is any tissue of a plant in itsnative state or in culture. This term includes, without limitation,whole plants, plant cells, plant organs, plant seeds, protoplasts,callus, cell cultures, and any group of plant cells organized intostructural and/or functional units. The use of this term in conjunctionwith, or in the absence of, any specific type of plant tissue as listedabove or otherwise embraced by this definition is not intended to beexclusive of any other type of plant tissue.

As used herein, the term “plant transformation vector” is a plasmid orviral vector that is capable of transforming plant tissue such that theplant tissue contains and expresses DNA that was not pre-existing in theplant tissue.

As used herein, the term “polypeptide” means a chain of at least twoamino acids joined by peptide bonds. The chain may be linear, branched,circular or combinations thereof. Preferably, polypeptides are fromabout 10 to about 1000 amino acids in length, more preferably 10-50amino acids in length. The polypeptides may contain amino acid analogsand other modifications, including, but not limited to glycosylated orphosphorylated residues.

As used herein, the term “recombinant polynucleotide” refers to apolynucleotide that has been altered, rearranged or modified by geneticengineering. Examples include any cloned polynucleotide, andpolynucleotides that are linked or joined to heterologous sequences. Twopolynucleotide sequences are heterologous if they are not naturallyfound joined together. The term recombinant does not refer toalterations to polynucleotides that result from naturally occurringevents, such as spontaneous mutations.

As used herein, the term “RNA” means ribonucleic acid and the term“mRNA” means messenger RNA.

As used herein, the term “transgenic” refers to any cell, tissue, organor organism that contains all or part of at least one recombinantpolynucleotide. In many cases, all or part of the recombinantpolynucleotide is stably integrated into a chromosome or stableextra-chromosomal element, so that it is passed on to successivegenerations.

Use of the term “transgenic” also refers to foreign polynucleotides. Useof the term “foreign” in this context refers to polynucleotides that mayor may not have been altered, rearranged or modified or even that havealso preexisted within the cell, tissue, organ or organism. Asillustration, a foreign polynucleotide is any polynucleotide that isintroduced or re-introduced into a cell, tissue, organ or organismregardless of whether the same or a similar polynucleotide has alreadypreexisted there. Any addition of any polynucleotide to a cell, tissue,organ or organism is considered by the present invention to beencompassed by the term “transgenic.”

As used herein, the term “transgenic plant” refers to any plant, plantcell, callus, plant tissue or plant part that contains all or part of atleast one recombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

As used herein, the term “transgenic plant material” is any plantmatter, including, but not limited to cells, protoplasts, tissues,leaves, seeds, stems, fruits and tubers both natural and processed,containing a recombinant polynucleotide. Further, plant materialincludes processed derivatives thereof including, but not limited tofood products, food stuffs, food supplements, extracts, concentrates,pills, lozenges, chewable compositions, powders, formulas, syrups,candies, wafers, capsules and tablets.

The terms “reduced expression”, “suppressed expression” or “knock out”,which are used interchangeably herein, are intended to mean that theexpression of a target sequence is suppressed over the expression ofthat sequence that is observed in conventional transgenic lines forheterologous sequences or over endogenous levels of expression forhomologous sequences. Heterologous or exogenous sequences comprisesequences that do not occur in the plant of interest in its nativestate. Homologous or endogenous sequences are those that are nativelypresent in the plant genome. Generally, expression of the targetsequence is reduced at least about 10%, preferably about 30%, morepreferably about 50%, and even more preferably about 80% and a greaterdegree of reduction of expression is yet more preferable. The methods ofthe invention provide for a substantial reduction in expression.However, it is not required that the reduction in expression have aneffect on the plant that is observable by visual inspection.

Sequence relationships between two or more nucleic acids orpolynucleotides are generally defined as sequence identity, percentageof sequence identity, and substantial identity. In determining sequenceidentity, a “reference sequence” is used as a basis for sequencecomparison. The reference sequence may be a subset or the entirety of aspecified sequence. That is, the reference sequence may be a full-lengthgene sequence or a segment of the gene sequence.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or two polypeptides, refers to two or more sequences orsubsequences that have at least about 50% nucleotide or amino acidresidue identity when compared and aligned for maximum correspondence asmeasured using one of the following sequence comparison algorithms or byvisual inspection. In preferred embodiments, substantially identicalsequences have at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, and even more preferably haveabout 90% or 95% nucleotide or amino acid residue identity. Preferably,the substantial identity exists over a region of the sequences that isat least about 50 residues in length, more preferably over a region ofat least about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical whenthey are identical over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410 (1990), and Altschul et al., Nucleic Acids Res. 25: 3389-3402(1977), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid. Thus, a polypeptide is typicallysubstantially identical to a second polypeptide, for example, where thetwo peptides differ only by conservative substitutions. Anotherindication that two nucleic acid sequences are substantially identicalis that the two molecules hybridize to each other under stringentconditions, as described below.

As discussed above, sequence identity, or identity, in the context ofnucleic acid or polypeptide sequences refers to the nucleic acid basesor residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. “Percentageof sequence identity” refers to the value determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions as compared to the reference window foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

Nucleotide sequences are generally substantially identical if the twomolecules hybridize to each other under stringent conditions. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point for the specific sequence at a defined ionicstrength and pH. Nucleic acid molecules that do not hybridize to eachother under stringent conditions may still be substantially identical ifthe polypeptides they encode are substantially identical. This can occurwhen a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

As noted, hybridization of sequences may be carried out under stringentconditions. By “stringent conditions” is intended conditions under whicha probe will hybridize to its target sequence to a detectably greaterdegree than to other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1.0 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. It is recognized that the temperature salt andwash conditions may be altered to increase or decrease stringencyconditions. For the post-hybridization washes, the critical factors arethe ionic strength and temperature of the final wash solution. See,Meinkoth and Wahl, Anal. Biochem. 138:267-284 (1984).

As indicated, fragments and variants of the peptide and nucleotidesequences of the invention are encompassed herein. By “fragment” isintended a portion of the sequence. In the case of a nucleotidesequence, fragments of the enhancer sequence will generally retain thebiological activity of the native suppressor protein. Alternatively,fragments of the targeting sequence may or may not retain biologicalactivity. Such targeting sequences may be useful as hybridizationprobes, as antisense constructs, or as co-suppression sequences. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence of the invention.

The term “variants” means substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of the enhancer of the invention. Variant nucleotidesequences include synthetically derived sequences, such as thosegenerated, for example, using site-directed mutagenesis. Generally,nucleotide sequence variants of the invention will have at least 40%,50%, 60%, 70%, generally 80%, preferably 85%, 90%, up to 95%, 98%sequence identity to its respective native nucleotide sequence.

Variant suppressor or enhancer proteins may also be utilized. A“variant” protein is intended to include a protein derived from thenative protein by deletion or addition of one or more amino acids to theN-terminal and/or C-terminal end of the native protein; deletion oraddition of one or more amino acids at one or more sites in the nativeprotein; or substitution of one or more amino acids at one or more sitesin the native protein. Such variants may result from, for example,genetic polymorphism or human manipulation. Conservative amino acidsubstitutions will generally result in variants that retain biologicalfunction.

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning-ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, JohnWiley & Sons, Inc. (1994-1998).

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention inserted into any vector capable of delivering thenucleic acid molecule into a cell. Such a vector contains heterologousnucleic acid sequences, that is, nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, and can be either prokaryotic or eukaryotic.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to control sequences within an expression vector. Thephrase “operatively linked” refers to insertion of a nucleic acidmolecule into an expression vector in a manner such that the molecule isable to be expressed when transformed into a cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga cell and of effecting expression of a specified nucleic acid molecule.Preferably, the expression vector is also capable of replicating withinthe cell. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, yeast,crustacean, mammalian, insect, other animal, and plant cells.

In preferred embodiments, expression vectors of the present inventioncontain regulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences that control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those that control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art.

The transcriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

Where appropriate, the RAV gene(s) for modulating RNA silencing may beoptimized for expression in the transformed plant. That is, the genescan be synthesized using plant-preferred codons corresponding to theplant of interest. Methods are available in the art for synthesizingplant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and5,436,391, and Murray et al., Nucleic Acids Res. 17:477-498 (1989).

Preferred expression vectors of the present invention can direct ntRAVoverexpression or suppression in plant cells.

The present invention also provides vectors containing the expressioncassettes of the invention. By “vector”, what is meant is apolynucleotide sequence that is able to replicate in a host cell, butsuch definition also includes transient vectors that could be used tointroduce DNA by, for example, homologous recombination in the genome,which do not replicate in a host cell. The vector can comprise DNA orRNA and can be single or double stranded, and linear or circular.Various plant expression vectors and reporter genes are described inGruber et al. in Methods in Plant Molecular Biology and Biotechnology,Glick et al., eds, CRC Press, pp. 89-119 (1993); and Rogers et al., MethEnzymol 153:253-277 (1987). Standard techniques for the construction ofthe vectors of the present invention are well-known to those of ordinaryskill in the art and can be found in such references as Sambrook et al.,Ibid.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. These vectors are listed solely by way of illustrationof the many commercially available and well known vectors that areavailable to those of skill in the art for use in accordance with thisaspect of the present invention. It will be appreciated that any otherplasmid or vector suitable for, for example, introduction, maintenance,propagation or expression of a polynucleotide or polypeptide of theinvention in any host cell or tissue may be used in this aspect of theinvention.

The vectors of the invention can contain 5′ and 3′ regulatory sequencesnecessary for transcription and termination of the polynucleotide ofinterest. Thus, the vectors can include a promoter and a transcriptionalterminator.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al., PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., MDMV leader (Maize Dwarf MosaicVirus); Virology 154:9-20 (1986)), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al., Nature, 353:90-94 (1991));untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al., Nature 325:622-625 (1987)); tobacco mosaicvirus leader (TMV) (Gallie et al. in Molecular Biology of RNA, ed. Cech(Liss, N.Y.), pp. 237-256 (1989)): and maize chlorotic mottle virusleader (MCMV) (Lommel et al., Virology 81:382-385 (1991)). See also,Della-Cioppa et al., Plant Physiol. 84:965-968 (1987).

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Other functional sequences may be included in the vectors of theinventions. Such functional sequences include, but are not limited to,introns, enhancers and translational initiation and termination sitesand polyadenylation sites. Preferably, the expression cassettes of thepresent invention are engineered to contain a constitutive promoter 5′to its translation initiation codon (ATG) and a poly(A) addition signal(AATAAA) 3′ to its translation termination codon. The control sequencescan be those that can function in at least one plant, plant cell, planttissue, or plant organ. These sequences may be derived from one or moregenes, or can be created using recombinant technology.

Other elements such as introns, enhancers, polyadenylation sequences andthe like may also be a part of the recombinant DNA. Such elements may ormay not be necessary for the function of the DNA, but may provideimproved expression of the mRNA by affecting transcription, stability,or the like. For example, the maize AdhIS first intron may be placedbetween the promoter and the coding sequence in a particular recombinantDNA construction. This intron, when included in a DNA construction, isknown to increase production of a protein in maize cells. (J. Callis etal., Genes and Develop., 1:1183 (1987)). However, sufficient expressionfor a selectable marker to perform satisfactorily can often be obtainedwithout an intron. See T. Klein et al., Plant Physiol., 91:440 (1989).An example of an alternative suitable intron is the shrunken-I firstintron of Zea mays.

The 5′ regulatory sequences (promoters) which are often used in creationof chimeric genes for plant transformation may cause either nominallyconstitutive expression in all cells of the transgenic plant, orregulated gene expression where only specific cells, tissues, or organsshow expression of the introduced genes.

As used herein, the terms “promoter” or “regulatory DNA sequence” meansan untranslated DNA sequence which assists in, enhances, or otherwiseaffects the transcription, translation or expression of an associatedstructural DNA sequence which codes for a protein or other DNA product.The promoter DNA sequence is usually located at the 5′ end of atranslated DNA sequence, typically between 20 and 100 nucleotides fromthe 5′ end of the translation start site.

Promoters useful in the expression cassettes of the invention includeany promoter that is capable of initiating transcription in a cell.Promoters which are known or found to cause transcription of a foreigngene in plant cells can be used in the present invention. Such promotersmay be obtained from plants, bacteria, animals or viruses and include,but are not necessarily limited to the constitutive 35S promoter ofcauliflower mosaic virus (CaMV) (as used herein, the phrase “CaMV 35S”promoter, includes variations of CaMV 35S promoter such as the 2×CaMV35S promoter). See Odell et al., Nature 313:810-812 (1985). Constitutivepromoters such as the 35S promoter are active under most conditions.

Examples of constitutive promoters that are useful in the presentinvention include the Sep1 promoter, the rice actin promoter (McElroy etal., Plant Cell 2:163-171 (1990)), the Arabidopsis actin promoter, theubiquitan promoter (Christensen et al., Plant Molec Biol 18:675-689(1989)); the pEmu promoter (Last et al., Theor Appl Genet 81:581-588(1991)), the figwort mosaic virus 35S promoter, the Smas promoter(Velten et al., EMBO J. 3:2723-2730 (1984)), the GRP1-8 promoter, thecinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),promoters from the T-DNA of Agrobacterium, such as those from themannopine synthase, nopaline synthase, and octopine synthase genes, thesmall subunit of ribulose bisphosphate carboxylase (ssuRUBISCO)promoter, and the like. Other constitutive promoters include those inU.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785;5,399,680; 5,268,463; and 5,608,142.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as fruits, leaves, roots, stems, seeds, or xylem. Examplesof tissue preferred and organ preferred promoters include, but are notlimited to Prha (expressed in root, seedling, lateral root, shoot apex,cotyledon, petiole, inflorescence stem, flower, stigma, anthers, andsilique, and auxin-inducible in roots); VSP2 (expressed in flower buds,flowers, and leaves, and wound inducible); SUC2 (expressed in vasculartissue of cotyledons, leaves and hypocotyl phloem, flower buds, sepalsand ovaries); AAP2 (silique-preferred); SUC1 (anther and pistilpreferred); AAP1 (seed preferred); Saur-AC1 (auxin inducible incotyledons, hypocotyl and flower); Enod 40 (expressed in root, stipule,cotyledon, hypocotyl and flower); and VSP1 (expressed in young siliques,flowers and leaves).

The present invention may utilize promoters for genes which are known togive high-level expression in edible plant parts, such as thetuber-specific patatin gene promoter from potato. For furtherinformation, see, e.g., H. C. Wenzler, et al, Plant Mol. Biol., 12:41-50(1989). It is also known that a tissue-specific promoter for one speciesmay be used successfully in directing transgenic DNA expression inspecific tissues of other species. For example, the potato tuberpromoters will also function in tomato plants to cause fruit specificexpression of an introduced gene. See U.S. patent Ser. No. 08/344,639,to Barry, et al.

A further example of a tissue-specific promoter is the seed specificbean phaseolin and soybean beta-conglycinin promoters. See, e.g., Keeleret al., Plant. Mol. Biol. 34:15-29 (1997).

Examples of other promoters for use with an embodiment of the presentinvention include fruit-specific promoters such as the E8 promoter,described in Deikman et al., EMBO J. 2:3315-3320 (1998). The activity ofthe E8 promoter is not limited to tomato fruit, but is thought to becompatible with any system wherein ethylene activates biologicalprocess, including fruit ripening. See U.S. Pat. No. 5,234,834 toFischer et al. Other promoters suitable for use with the presentinvention are the MADS-box promoters, endo-β-1,4-glucanase promoter,expansin promoters, egase promoters, pectate lyase promoter,polygalacturonase promoters, and ethylene biosynthesis promoters.

Also useful in the present invention are: (a) another tomatofruit-specific promoter, LeExp-1 (See U.S. Pat. No. 6,340,748); (b) thebanana fruit-specific promoters known as the MT clones (See U.S. Pat.No. 6,284,946); and (c) several strawberry specific promoters termedGSRE2, GSRE49, SEL1, and SEL2 (See U.S. Pat. No. 6,080,914).

Leaf-specific promoters include, Yamamoto et al., Plant J. 12(2):255-265(1997); Kwon et al., Plant Physiol. 105:357-67 (1994); Yamamoto et al.,Plant Cell Physiol. 35(5):773-778 (1994); Gotor et al., Plant J.3:509-18 (1993); Orozco et al., Plant Mol. Biol. 23(6): 1129-1138(1993); and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90(20):9586-9590(1993).

Root-specific promoters are known and can be selected from the manyavailable from the literature. See, for example, Hire et al., Plant Mol.Biol. 20(2): 207-218 (1992) (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, Plant Cell 3(10):1051-1061 (1991)(root-specific control element in the GRP 1.8 gene of French bean);Sanger et al., Plant Mol. Biol. 14(3):433-443 (1990) (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens); Miao et al., Plant Cell 3(1):11-22 (1991) (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). Bogusz et al., PlantCell 2(7):633-641 (1990) (root-specific promoters from hemoglobin genesfrom the nitrogen-fixing nonlegume Parasponia andcersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa). Leach andAoyagi, Plant Science (Limerick) 79(1):69-76 (1991) (roIC and roIDroot-inducing genes of Agrobacterium rhizogenes); Teeri et al., EMBO J.8(2):343-350 (1989) (octopine synthase and TR2′ gene); (VfENOD-GRP3 genepromoter); Kuster et al., Plant Mol. Biol. 29(4):759-772 (1995) andCapana et al., Plant Mol. Biol. 25(4):681-691 (1994) roIB promoter. Seealso U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

Anther or pollen-specific promoters may be used to create male sterileplants. While either the targeting sequence or the enhancer may beoperably linked to such promoters, it may be preferred to express boththe enhancer and the targeting sequence with an anther specific orpollen specific promoter to prevent even low expression of the toxin inother tissues of the plant.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al.,BioEssays 10:108 (1989). Such seed-preferred promoters include, but arenot limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); ceIA (cellulose synthase); gama-zein; Glob-1; bean.beta.-phaseolin; napin; .beta.-conglycinin: soybean lectin: cruciferin;maize 15 kDa zein; 22 kDa zein; 27 kDa zein; g-zein; waxy; shrunken 1;shrunken 2; globulin 1, etc.

A number of inducible promoters are known in the art, which can be usedin conjunction with the present invention. For resistance genes, apathogen-inducible promoter can be utilized. Such promoters includethose from pathogenesis-related proteins (PR proteins), which areinduced following infection by a pathogen, e.g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase, and the like. See, for example,Redolfi et al., Neth. J. Plant Pathol. 89:245-254 (1983); Uknes et al.,Plant Cell 4:645-656 (1992); and Van Loon, Plant Mol. Virol. 4:111-116(1985). Of particular interest arc promoters that are expressed locallyat or near the site of pathogen injection. See, for example, Marineau etal., Plant Mol. Biol. 9:335-342 (1987); Matton et al., MolecularPlant-Microbe Interactions 2:325-331 (1989); Somsisch et al., Proc.Natl. Acad. Sci. USA 83:2427-2430 (1986): Somsisch et al., Mol. Gen.Genet., 2:93-98 (1988); and Yang, Proc. Natl. Acad. Sci. USA93:14972-14977 (1996). See also, Chen et al., Plant J. 10:955-966(1996); Zhang et al., Proc. Natl. Acad. Sci. USA 91:2507-2511 (1994);Warner et al., Plant J., 3:191-201 (1993): Siebertz et al, Plant Cell1:961-968 (1989): U.S. Pat. No. 5,750,386; and Cordero et al., Physiol.Mol. Plant. Path. 41:189-200 (1992).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in the DNAconstructs of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan, Ann. Rev. Phytopath.28:425-449 (1990); Duan et al., Nature Biotechnology 14:494-498 (1996);wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al,Mol. Gen. Genet. 215:200-208 (1989)); systemin (McGurl et al., Science225:1570-1573 (1992)); WIP1 (Rohmeier et al., Plant Mol. Biol.22:783-792 (1993); Eckelkamp et al., FEBS Letters 323:73-76 (1993); MPIgene (Corderok et al., Plant J. 6(2):141-150 (1994)); and the like.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1 a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al., Proc. Natl. Acad. Sci. USA 88:10421-10425 (1991), andMcNellis et al., Plant J. 14(2):247-257 (1998), andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al., Mol. Gen. Genet. 227:229-237 (1991), and U.S. Pat.Nos. 5,814,618 and 5,789,156).

The recombinant polynucleotides that are to be introduced into the plantcells will preferably contain either a selectable marker or a reportergene, or both, in order to facilitate identification and selection oftransformed cells. Alternatively, the selectable marker can be carriedon a separate piece of DNA and used in a co-transformation procedure.Both selectable markers and reporter genes can be flanked withappropriate regulatory sequences to enable expression in plants. Usefulselectable markers are well known in the art and include, for example,antibiotic and herbicide resistance genes. Examples of such markersinclude genes encoding drug or herbicide resistance, such as hygromycinresistance (hygromycin phosphotransferase (HPT)), spectinomycinresistance (encoded by the aada gene), kanamycin and gentamycinresistance (neomycin phosphotransferase (nptII)), streptomycinresistance (streptomycin phosphotransferase gene (SPT)),phosphinothricin or basta resistance (barnase (bar)), chlorsulfuronresistance (acetolactase synthase (ALS)), chloramphenicol resistance(chloramphenicol acetyl transferase (CAT)), G418 resistance, lincomycinresistance, methotrexate resistance, glyphosate resistance, and thelike.

The expression cassettes of the invention may be covalently linked togenes encoding enzymes that are easily assayed, for example, luciferase,alkaline phosphatase, B-galactosidase (B-gal), B-glucuronidase (GUS),and the like.

Transcriptional termination regions include, but are not limited to, theterminators of the octopine synthase and nopaline synthase genes in theA. tumefaciens Ti plasmid. For further information, see Ballas et al.,Nuc Acid Res 17:7891-7903 (1989). If translation of the transcript isdesired, translational start and stop codons can also be provided.

Plants suitable for transformation according to the processes of thisinvention include, without limitation, both dicotyledon andmonocotyledon plant cells can be used as a host organism in the presentinvention. For example, the present invention may be used fortransformation of any plant species, including, but not limited to, corn(Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa(Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables,ornamentals, and conifers.

Preferably, plants of the present invention are crop plants (forexample, cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorghum, millet, cassava, barley, pea, and other root, tuber, or seedcrops. Important seed crops are oil-seed rape, sugar beet, maize,sunflower, soybean, and sorghum. Horticultural plants to which thepresent invention may be applied may include lettuce, endive, andvegetable brassicas including cabbage, broccoli, and cauliflower, andcarnations and geraniums. The present invention may be applied totobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum, poplar, eucalyptus, and pine.

Grain plants that provide seeds of interest include oil-seed plants andleguminous plants. Seeds of interest include grain seeds, such as corn,wheat, barley, rice, sorghum, rye, etc. Oil seed plants include cotton,soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,etc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava bean, lentils, chickpea, etc.

Plants transformed with a DNA construct of the invention may be producedby standard techniques known in the art for the genetic manipulation ofplants. The particular choice of a transformation technology will bedetermined by its efficiency to transform certain plant species as wellas the experience and preference of the person practicing the inventionwith a particular methodology of choice. It will be apparent to theskilled person that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

For example, various methods have been successfully demonstrated totransform plants in a stable manner. Such methods include, among others;Agrobacterium-mediated transformation, viral-mediated transformation,biolistics (microprojectile bombardment), protoplasts (PEG),electroporation, and microinjection. A typical procedure involves themechanical, bacterial, or viral introduction of the foreign DNA, whichencodes for the biosynthetic pathway or protein of interest, intoimmature plant parts. As the “transformed” immature plant develops, theforeign DNA becomes incorporated into the plant's own DNA.

Methods and compositions for transforming a bacterium, a fungal cell, aplant cell, or an entire plant with one or more expression vectorscomprising an ntRAV protein-encoding gene segment are further aspects ofthis disclosure. A transgenic bacterium, fungal cell (such as, forexample, a yeast cell), plant cell or plant derived from such atransformation process or the progeny and seeds from such a transgenicplant are also further embodiments of the invention. A variety oftechniques are available for the introduction of the genetic materialinto or transformation of the plant cell host. However, the particularmanner of introduction of the plant vector into the host is not criticalto the practice of the present invention, and any method which providesfor efficient transformation can be employed.

Methods for the introduction of polynucleotides into plants and forgenerating transgenic plants are known to those skilled in the art. Seee.g. Weissbach & Weissbach, Methods for Plant Molecular Biology,Academic Press, N.Y.; Grierson & Corey (1988).

The choice of plant tissue source or cultured plant cells fortransformation will depend on the nature of the host plant and thetransformation protocol. Useful tissue sources include callus,suspension culture cells, protoplasts, leaf segments, root segments,stem segments, tassels, pollen, embryos, hypocotyls, tuber segments,meristematic regions, and the like. In preferred embodiments, the tissuesource is regenerable, in that it will retain the ability to regeneratewhole, fertile plants following transformation.

In a preferred embodiment of the present invention, the Agrobacterium-Tiplasmid system is utilized. Agrobacterium tumefaciens-meditatedtransformation techniques are well described in the scientificliterature. See, e.g., Horsch et al., Science 233: 496-498 (1984).Descriptions of the Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided in Gruber et al.,supra. Although Agrobacterium is useful primarily in dicots, certainmonocots can also be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Agrobacterium-mediated transformation utilizes A. tumefaciens, theetiologic agent of crown gall, a disease of a wide range of dicotyledonsand gymnosperms that results in the formation of tumors or galls inplant tissue at the site of infection. Agrobacterium, which normallyinfects the plant at wound sites, carries a large extrachromosomalelement called Ti (tumor-inducing) plasmid.

Ti plasmids contain two regions required for tumor induction. One regionis the T-DNA (transfer-DNA), which is the DNA sequence that isultimately found stably transferred to plant genomic DNA. The otherregion is the vir (virulence) region, which has been implicated in thetransfer mechanism. Although the vir region is absolutely required forstable transformation, the vir DNA is not actually transferred to theinfected plant. Transformation of plant cells mediated by infection withA. tumefaciens and subsequent transfer of the T-DNA alone have been welldocumented. See, e.g., Bevan, M. W. et al., Int. Rev. Genet, 16 357(1982).

The construction of an Agrobacterium transformation vector system hastwo elements. First, a plasmid vector is constructed which replicates inEscherichia coli (E. coli). This plasmid contains the DNA encoding theprotein of interest (in this invention an over-expressed or knocked-outntRAV transgene). This DNA is flanked by T-DNA border sequences thatdefine the points at which the DNA integrates into the plant genome.Border sequences include both a left border (LB) and a right border(RB).

Usually a gene encoding a selectable marker (such as a gene encodingresistance to an antibiotic or herbicide such as hygromycin or Basta) isalso inserted between the left border and right border sequences. Theexpression of this gene in transformed plant cells gives a positiveselection method to identify those plants or plant cells which have anintegrated T-DNA region. The second element of the process is totransfer the plasmid from E. coli to Agrobacterium. This can beaccomplished via a conjugation mating system, or by direct uptake ofplasmid DNA by Agrobacterium using such methods as electroporation.

Those skilled in the art would recognize that there are multiple choicesof Agrobacterium strains and plasmid construction strategies that can beused to optimize genetic transformation of plants. They will alsorecognize that A. tumefaciens may not be the only Agrobacterium strainused. Other Agrobacterium strains such as A. rhizogenes might be moresuitable in some applications. See Lichtenstein and Fuller in: GeneticEngineering, Volume 6, Ribgy (ed) Academic Press, London (1987).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, e.g., bombardmentwith Agrobacterium-coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

The present invention is not limited to the Agrobacterium-Ti plasmidsystem, and includes any direct physical method of introducing foreignDNA into an organism such as plant cells. Direct transformation involvesthe uptake of exogenous genetic material into cells or protoplasts. Suchuptake may be enhanced by use of chemical agents or electric fields. SeeDewulf J. and Negrutiu I., Direct gene transfer into protoplasts: Thechemical approach, in: A Laboratory Guide for Cellular and MolecularPlant Biology. Eds. I. Negrutiu and G. Gharti-Chhetri. BirkhauserVerlag. Basel (1991). The exogenous genetic material may then beintegrated into the nuclear genome. For mammalian cells, transfectionprocedures can be used. For bacterial cells, electroporation or heatshock methods can be employed.

For plant systems, direct gene transfer can also be accomplished bypolyethylene glycol (PEG) mediated transformation. This method relies onchemicals to mediate the DNA uptake by protoplasts and is based onsynergistic interactions between Mg⁺², PEG, and possibly Ca⁺². See,e.g., Negrutiu, R. et al., Plant Mol. Biol., 8: 363 (1987).Alternatively, exogenous DNA can be introduced into cells or protoplastsby microinjection. In this technique, a solution of the plasmid DNA orDNA fragment is injected directly into the cell with a finely pulledglass needle.

Another procedure for direct gene transfer involves bombardment of cellsby micro-projectiles carrying DNA. In this procedure, commonly calledparticle bombardment or biolistics, tungsten or gold particles coatedwith the exogenous DNA are accelerated toward the target cells. Theparticles penetrate the cells carrying with them the coated DNA.Microparticle acceleration has been successfully demonstrated to giveboth transient expression and stable expression in cells suspended incultures, protoplasts, immature embryos of plants, including, but notlimited to, onion, maize, soybean, and tobacco. Microprojectiletransformation techniques are described in Klein T. M., et al, Nature,327: 70-73 (1987).

Electric fields may also be used to introduce genetic material into thecells of an organism. The application of brief, high-voltage electricpulses to a variety of bacterial, animal and plant cells leads to theformation of nanometer-sized pores in the plasma membrane. DNA is takendirectly into the cell cytoplasm either through these pores or as aconsequence of the redistribution of membrane components thataccompanies closure of the pores. Electroporation techniques aredescribed in Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985).

Viral means of introducing DNA into cells are known in the art. Inparticular, a number of viral vector systems are known for theintroduction of foreign or native genes into mammalian cells. Theseinclude the SV40 virus (See, e.g., Okayama et al., Molec. Cell Biol.5:1136-1142 (1985)); bovine papilloma virus (See, e.g., DiMaio et al,Proc. Natl. Acad. Sci. USA 79:4030-4034 (1982)); adenovirus (See, e.g.,Morin et al., Proc. Natl. Acad. Sci. USA 84:4626 (1987)). For furtherinformation regarding viral vector systems, see, e.g., Yifan et al.,Proc. Natl. Acad. Sci. USA 92:1401-1405 (1995).

A number of viral vector systems are also known for the introduction offoreign or native genes into plant cells. A variety of plant virusesthat can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

Following transformation, a plant may be regenerated, e.g., from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues, and organs ofthe plant. Available techniques are reviewed in Vasil et al., in CellCulture and Somatic Cell Genetics of Plants, Vols. I, II, and III,Laboratory Procedures and Their Applications (Academic Press) (1984);and Weissbach et al., Methods For Plant Mol. Biol. (1989). Theparticular method of regeneration will depend on the starting planttissue and the particular plant species to be regenerated. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillilan Publishing Company, New York, pp. 124-176 (1983).

In recent years, it has become possible to regenerate many species ofplants from callus tissue derived from plant explants. Regeneration ofplants from tissue transformed with A. tumefaciens has been demonstratedfor several species of plants. These include, but are not limited to,sunflower, tomato, white clover, rapeseed, cotton, tobacco, potato,maize, rice, and numerous vegetable crops.

The transformed plants may then be grown, and either pollinated with thesame transformed strain or different strains, and the resulting hybridhaving expression of the desired phenotypic characteristic identified.Two or more generations may be grown to ensure that expression of thedesired phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

Likewise, conventional plant breeding methods can be used, including,but not limited to crossing and backcrossing, self-pollination andvegetative propagation. Techniques for breeding plants are known tothose skilled in the art.

The progeny of a transgenic plant are included within the scope of theinvention, provided that the progeny contain all or part of the ntRAVtransgenic construct. Thus, according to the invention there is provideda plant cell having the constructs of the invention. A further aspect ofthe present invention provides a method of making such a plant cellinvolving introduction of a vector including the construct into a plantcell. For integration of the construct into the plant genome, suchintroduction will be followed by recombination between the vector andthe plant cell genome to introduce the sequence of nucleotides into thegenome. RNA encoded by the introduced nucleic acid construct may then betranscribed in the cell and descendants thereof, including cells inplants regenerated from transformed material. A gene stably incorporatedinto the genome of a plant is passed from generation to generation todescendants of the plant, so such descendants should show the desiredphenotype.

The present invention also provides a plant comprising a plant cell asdisclosed. Transformed seeds and plant parts are also encompassed.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings, seed. The invention provides any plantpropagule that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed and so on. Alsoencompassed by the invention is a plant which is a sexually or asexuallypropagated off-spring, clone or descendant of such a plant, or any partor propagule of said plant, off-spring, clone or descendant. Plantextracts and derivatives are also provided.

The following examples describe embodiments of the invention. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples. In the examples, all percentages aregiven on a weight basis unless otherwise indicated.

EXAMPLE 1

This example shows the production of both an ntRAV over-expressedtobacco line and an ntRAV-silenced transgenic tobacco line.

Transgenic tobacco lines were produced that over-expressed ntRAV andwere silenced for ntRAV. Each of these lines were then crossed to atransgenic tobacco line (“6b5”) that is silenced in response to asense-transgene encoding beta-glucosidase (GUS). For example, transgenictobacco plants overexpressing ntRAV were generated by introducing atransgene into tobacco cells that expressed ntRAV cDNA under control ofthe 35S cauliflower mosaic virus promoter. Transgenic plants partiallysilenced for ntRAV were generated by introducing a transgene thatcontained the ntRAV sequences in an inverted repeat (dsRAV) to inducethe RNA silencing. Both the overexpressing and the knockdown transgenictobacco lines were phenotypically wild-type.

The 6b5 Tobacco line was generated by cloning the bacterial UidA genebetween the 35S promoter with a double enhancer and the terminatorsequences of the pea rbcS 9C gene and introduced into tobacco plants byagroinfiltration. Tobacco line X-27-8 is N. tabacum cv Xanthi NCtransformed with the wild-type P1/HC-Pro sequence. Line X-27-8 wasconstructed for the experiments reported here by transformation withAgrobacterium. Tobacco line ntRAV-OX was generated by cloning the fulllength ntRAV gene behind the 35S promoter of the pFGC-1008 plasmid andtransformed into tobacco with Agrobacterium. The double stranded knockdown construct was the same as the OX construct except that the initialfull-length ntRAV sequence was followed by another ntRAV in reverseorientation and separated by a portion of the UidA gene.

EXAMPLE 2

This example shows histochemical staining of the ntRAV over-expressedand silenced transgenic tobacco line.

Leaves were assayed for GUS activity as described but with minormodifications. For staining of GUS in tobacco leaf tissue the leaveswere partially abraded on the lower side by using carborundum, fixed for20 min in 90% acetone, vacuum infiltrated with a buffer containing 50 mMsodium phosphate (pH 7.2), 0.5 mM K₃Fe (CN)₆, 0.5 mM K₃Fe(CN)₆, and 1 mM5-bromo-4-chloro-3-indolylβ-D-glucuronide and incubated at 37 deg C.Leaf pieces were subsequently treated with 95% ethanol to removechlorophyll.

EXAMPLE 3

This Example shows RNA Northern Analysis of the ntRAV over-expressed andsilenced transgenic tobacco line.

Step 1: RNA Isolation

RNA isolation was carried out as follows. Tissues were frozen in liquidnitrogen and cells were disrupted in a mortar and pestle withoutallowing the tissue to thaw. Cells were then homogenized in buffer (0.1M LiCl, 0.1M Tris-HCl, 0.01M ethylene diamine tetra acetic acid and 1%sodium dodecyl sulfate) and phenol/chloroform extracted. The highmolecular weight RNA is precipitated in 2M LiCl, spun down, washed in75% EtOH, resuspended in H₂O and quantified. The low molecular weightRNA is EtOH precipitated, resuspended in a smaller volume (200 ul) andseparated from residual high molecular weight RNA and DNA in 10%polyethylene glycol. Enriched low molecular weight RNA is then EtOHprecipitated and resuspended for quantification.

Step 2: Northern Blotting

Equal volumes (10 ul/lane) of high molecular weight RNA werefractionated in a 1% agarose gel using formaldehyde (0.66%) gelelectrophoresis and blotted to Hybond N paper (Amersham Corp.) usingcapillary action as described by Maniatis (1982). But with the followingmodification. RNA samples were incubated in sample buffer containing 60%formamide at 85 deg. C. for 2 minutes and at 65 deg. C. for 10 minutesto insure denaturation of double stranded RNA. Equal volumes (15μg/lane) of low molecular weight RNA were fractionated with denaturingpolyacrylamide gel electrophoresis and blotted to Hybond N paper.Randomly labeled (+) strand RNA probes for mRNA hybridization and (−)strand RNA probes for siRNA hybridization were generated from PCRfragment templates via a transcription reaction using P32 alpha-labeledUTP.

Levels of ntRAV were measured by Northern Blot of samples taken fromleaves of wild type tobacco plants (WT), from tobacco plants thatoverexpressed ntRAV with a 35S promoter (ntRAV), and from the knockdownline of tobacco plants having reduced levels of ntRAV expression(dsRAV), at 24, 30 and 37 days post germination (P.G.). All plants werecultivated under the same conditions. FIG. 1 shows that initiation ofGUS silencing in the wild type 6b5 line (WT) occurreddevelopmentally—the transgene was expressed in young seedlings, butdeclined with the onset of silencing at around three weeks aftergermination. In contrast, levels of ntRAV in the transgenic plantsoverexpressing ntRAV (ntRAV) show high levels of ntRAV at all threesampling times, whereas the knockdown line of plants (dsRAV) show lowlevels of ntRAV at all sampling times.

The ectopic over-expression of ntRAV delays the developmental onset ofGUS silencing in leaves of the 6b5 line by about two weeks. However,once begun, the pattern of RNA silencing is similar in ntRAVover-expressing lines and wild-type controls, beginning in veins ofolder leaves and spreading into adjacent blade tissue.

FIG. 2 illustrates the effects of sense transgene silencing of GUS inthe wild type tobacco line (WT), wherein silencing is seen to occurdevelopmentally. Silencing resets each generation. Silencing initiatesduring development, begins in the vascular system, and spreadsthroughout the leaf.

The pattern of accumulation of endogenous ntRAV mRNA parallels that ofthe silenced GUS mRNA, high in young seedlings and rapidly declining attwo to three weeks after germination. Together, these data suggest thatntRAV encodes an endogenous suppressor of RNA silencing that acts earlyin development to block RNA silencing. Consistent with these data, it isbelieved that the onset of GUS silencing was accelerated in an ntRAVsilenced tobacco line.

FIG. 3 shows the effect of reducing ntRAV expression on sense transgenesilencing of the reporter gene GUS in tobacco transgenic line 6b5.Histochemical staining to localize GUS expression in plants at 24 daysafter germination is shown in the upper part of the figure. Shown at thefar left is a leaf from the 6b5 transgenic plant that also expressesHC-Pro, a viral suppressor of RNA silencing (HC-Pro X 6b5). This leaf isdark colored throughout because silencing of the GUS gene is blocked.The second leaf from the left shows a leaf from a 6b5 transgenic plantthat also over-expresses the ntRAV gene and is also suppressed for RNAsilencing (ntRAV X 6b5). The third leaf from the left is from the 6b5line in an otherwise wildtype background (WT X 6b5). This leaf shows theinitiation of GUS silencing in the veins of the leaf: thus, the veins ofthe plants are not dark colored in some places due to lack of GUSactivity. The last leaf on the right is from the 6b5 line that alsocarries a transgene that expresses double-stranded (ds) RNA for theendogenous ntRAV gene (dsRAV X 6b5). This leaf shows more extensivesilencing of the GUS transgene as indicated by more extensive loss ofcolor in the veins.

Molecular data from the same plants is shown in the lower part of thefigure. The level of GUS mRNA and GUS siRNAs is shown in thesense-transgene silenced 6b5 transgenic line at 7, 14 or 24 dayspost-germination in an otherwise wildtype background (wt) or in thepresence of the HC-Pro transgene (HC-Pro) or a transgene that producesdouble-stranded ntRAV RNA (dsRAV) and thereby induces silencing of thentRAV gene. In an otherwise WT background, GUS silencing in line 6b5begins between 14 and 24 days after germination as indicated by thereduced level of GUS mRNA and the simultaneous appearance of the GUSsiRNAs, which are diagnostic of RNA silencing. HC-Pro blocks the onsetof silencing and prevents siRNA accumulation. In contrast, the presenceof the dsRAV transgene accelerates GUS silencing as indicated by thereduced levels of GUS mRNA even at 7 days after germination and theappearance of GUS siRNAs by 14 days after germination.

An illustration of the effectiveness of RNA silencing in tobacco plantsis shown in FIG. 4, where silencing in a wild type 6b5 cross (wt x 6b5)is contrasted with silencing in a knockdown ntRAV plant (dsRAV x 6b5),and it can be seen that silencing proceeds from the bottom to the top ofthe plant, and is visually more advanced in the knockdown ntRAV plantthan in the wild type plant.

EXAMPLE 4

This example illustrates the effect on sense transgene silencing of aknockout of the RAV-2 gene in Arabidopsis thaliana.

FIG. 9 (left-most box) shows histochemical staining of an Arabidopsisseedling of a transgenic line called L1. The L1 line is an example of asense transgene silenced line which is silenced for the GUS gene. Atthis early stage of development, the silencing has just begun and theyounger leaves of the seedling are expressing slightly less GUS and aretherefore are a lighter blue color. In contrast, an L1 seedling which ismutant for the RAV-2 (At1 g68840) gene due to a T-DNA insertion withinthat gene shows a much more advanced silencing at the same developmentalstage (FIG. 9, right-most box). Thus, a knock-out of the Arabidopsisthaliana RAV-2 gene enhances sense transgene silencing.

All references cited in this specification, including without limitationall papers, publications, patents, patent applications, presentations,texts, reports, manuscripts, brochures, books, internet postings,journal articles, periodicals, and the like, are hereby incorporated byreference into this specification in their entireties. The discussion ofthe references herein is intended merely to summarize the assertionsmade by their authors and no admission is made that any referenceconstitutes prior art. Applicants reserve the right to challenge theaccuracy and pertinency of the cited references.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results obtained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense. In addition, it should beunderstood that aspects of the various embodiments may be interchangedboth in whole or in part.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, or a sequence that is substantially identical thereto.2. The isolated nucleic acid molecule according to claim 1, comprisingthe nucleotide sequence of SEQ ID NO:1, or a sequence that is at leastabout 90% identical thereto.
 3. The isolated nucleic acid moleculeaccording to claim 1, comprising the nucleotide sequence of SEQ ID NO:1.4. A chimeric gene comprising the nucleotide sequence of SEQ ID NO:1, ora sequence that is substantially identical thereto, operably linked tosuitable regulatory sequences.
 5. A recombinant vector fortransformation of plant cells, the vector comprising a polynucleotidehaving the nucleotide sequence of SEQ ID NO:1, or a sequence that issubstantially identical thereto.
 6. A plant cell comprising anintroduced nucleotide sequence according to SEQ ID NO:1, or a sequencethat is substantially identical thereto.
 7. A plant comprising anintroduced nucleotide sequence according to SEQ ID NO:1, or a sequencethat is substantially identical thereto.
 8. A plant seed comprising anintroduced nucleotide sequence according to SEQ ID NO:1, or a sequencethat is substantially identical thereto.
 9. An isolated proteincomprising the amino acid sequence of SEQ ID NO:2, or a sequence that issubstantially identical thereto.
 10. The isolated protein according toclaim 9, comprising the amino acid sequence of SEQ ID NO:2, or asequence that is at least about 90% identical thereto.
 11. The isolatedprotein according to claim 9, comprising the amino acid sequence of SEQID NO:2.
 12. An antibody that binds with a protein comprising the aminoacid sequence of SEQ ID NO:2, or a sequence that is substantiallyidentical thereto, or binds with a polynucleotide comprising the nucleicacid sequence of SEQ ID NO:1, or a sequence that is substantiallyidentical thereto.
 13. A method of regulating gene silencing in a plantcell, the method comprising modulating in the plant cell the amount ofand/or the activity of a RAV protein.
 14. The method according to claim13, comprising increasing the amount of the protein in the plant cellby: a) introducing a gene encoding a RAV protein into the plant cell ina location where the gene is expressed; b) in a plant cell having anendogenous gene encoding a RAV protein, wherein the plant cell includesan endogenous promoter controlling the expression of the gene, insertinga heterologous promoter in a location that is operably linked the geneencoding the RAV protein, wherein the heterologous promoter drivesexpression of the gene to a degree that is greater than the endogenouspromoter; or c) adding RAV protein to the plant cell.
 15. The methodaccording to claim 13, comprising increasing the amount of the proteinin the plant cell by: a) introducing into the plant cell a genecomprising the nucleotide sequence of SEQ ID NO:1, or a sequence that issubstantially identical thereto, in a location where the gene isexpressed; b) in a plant cell having an endogenous gene comprising SEQID NO:1, or a nucleotide sequence that is substantially identicalthereto, wherein the plant cell includes an endogenous promotercontrolling the expression of the gene, inserting a heterologouspromoter in a location that is operably linked to SEQ ID NO: 1, or thesequence that is substantially identical thereto, wherein theheterologous promoter drives expression of the gene to a degree that isgreater than the endogenous promoter; or c) adding to the plant cell aprotein comprising the amino acid sequence of SEQ ID NO:2, or an aminoacid sequence that is substantially identical thereto.
 16. The methodaccording to part (b) of claim 15, where the heterologous promoter is aconstitutive promoter.
 17. The method according to part (a) of claim 15,comprising introducing into the plant cell a recombinant expressioncassette comprising a heterologous promoter sequence operably linked toa nucleotide sequence comprising SEQ ID NO:1, or a sequence that issubstantially identical thereto, wherein the promoter drives expressionof the polynucleotide sequence.
 18. The method according to claim 13, ina plant cell having an endogenous gene encoding a RAV protein, themethod comprising decreasing the amount of the RAV protein in the plantcell by: a) creating a knock out plant cell which has no or reducedexpression of the gene encoding a RAV protein; or b) introducing intothe plant cell a polynucleotide comprising a disrupted RAV gene, whereinthe polynucleotide has sufficient complementary to the endogenous RAVgene so that when the disrupted polynucleotide is introduced into thehost cell, it homologously recombines with the endogenous RAV gene andthereby reduces transcription of the endogenous gene.
 19. The methodaccording to claim 13, in a plant cell having an endogenous genecomprising SEQ ID NO:1, or a sequence that is substantially identicalthereto, the method comprising decreasing the amount of the protein inthe plant cell by: a) creating a knock out plant cell which has no orreduced expression of the gene; or b) introducing into the plant cell apolynucleotide comprising a disrupted SEQ ID NO.1, wherein thepolynucleotide has sufficient complementary to the endogenous gene sothat when the disrupted polynucleotide is introduced into the host cell,it homologously recombines with the endogenous gene and thereby reducestranscription of the endogenous gene.
 20. The method according to claim13, comprising decreasing in the plant cell the activity of a RAVprotein by adding to the cell or causing the cell to produce compoundsthat inhibit the activity of the RAV protein.
 21. The method accordingto claim 13, comprising decreasing in the plant cell the activity of aprotein that includes an amino acid sequence according to SEQ ID NO: 2,or a fragment thereof, or a protein that is substantially identicalthereto by adding to the cell or causing the cell to produce compoundsthat inhibit the activity of the protein.
 22. The method according toclaim 20, comprising adding to the cell or causing the cell to producean antibody that is specific for the protein.
 23. The method accordingto claim 13, wherein the method is carried out on plant cells in vitro.24. The method according to claim 13, wherein the method is carried outon plant cells in vivo.
 25. The method according to part (a) of claim15, wherein the amount of the protein in the plant cell is increased byintroducing into the plant cell a gene comprising the nucleotidesequence of SEQ ID NO:1, or a sequence that is substantially identicalthereto, and further comprising regenerating a whole plant from theplant cell.
 26. A method of regulating post transcriptional genesilencing in a plant, the method comprising: (a) controlling in theplant the expression of a gene that encodes a RAV protein; or (b)controlling in the plant the amount of and/or the activity of a RAVprotein.
 27. The method according to claim 26, comprising: (a)controlling in the plant the expression of a gene that includes anucleotide sequence according to SEQ ID NO: 1, or a sequence that issubstantially identical thereto; or (b) controlling in the plant theamount of and/or the activity of a protein that includes an amino acidsequence according to SEQ ID NO: 2, or an amino acid sequence that issubstantially identical thereto, and which protein has the samebiological activity as a protein that includes an amino acid sequenceaccording to SEQ ID NO: 2.